1. Field of the Invention
The present invention relates to a dry immunoassay element in which a homogeneous enzyme immunoassay is utilized, and an immunoassaying process in which the dry immunoassay element is used.
Analyses of the constituents originated from the living body or chemicals contained in the body fluids, such as blood and urine, are useful for diagnosing the condition of diseases or judging the course of curing, and thus they occupy important parts in the field of clinical test. The so-called enzyme immunoassay has been known in the art as one method for analyzing such constituents (ligands) generally present in a small amount in the body fluids. The enzyme immunoassay may be classified into heterogeneous systems for which B/F (Bound/Free) separation must be effected, and homogeneous system for which B/F separation is not necessary. The reactions in the homogeneous system are based on the phenomenon that the enzymatic activity of the labelling enzyme is affected by some interference caused by binding of an antibody to the antigen (ligand), and the inhibition due to antigen-antibody binding is generally utilized. It is considered that the enzymatic activity is suppressed by a steric hindrance caused by binding the enzyme to the substrate or a change in three-dimensional structure of the enzyme, when the antibody which is generally a large molecule is bound to the antigen in the enzyme-labelled antigen.
When the antigen is a high polymer, suppression of enzymatic activity by the antigen-antibody binding reaction may be detected by labelling the antibody with an enzyme.
Meanwhile, in the routine clinical tests in which a number of test samples are to be handled, it is demanded that the individual samples should be analyzed by simple operations, more desirously by automated operation sequence.
2. Prior Art Statement
To comply with the demand, dry analysis elements have been proposed (see, for example, Unexamined Japanese Patent Publication Nos. 53888/1974 (corresponding to U.S. Pat. No. 3,992,158), 77356/1984 (corresponding to EP 0097952A) and 102388/1984 and U.S. Pat. No. 4,459,358.)
A dry analysis element has been known, in which an enzyme-labelled antibody is utilized and reacted in a homogeneous enzyme immunological reaction (see Unexamined Japanese Patent Publication No. 321360/1989 which corresponds to EP 0347839A). This known dry analysis element comprises the following three reagent ingredients in the same or different layers in the composite multi-layered structure:
(A) An antigen having a high molecular weight (a coupling product of a ligand or a derivative thereof with a high molecular weight compound; hereinafter referrred to as "polymerized antigen"); PA1 (B) A water-insoluble high polymer substrate; and PA1 (C) A conjugate of an antibody against the ligand and an enzyme for the substrate. PA1 (A) Water-insoluble high polymer substrate; and PA1 (B) Conjugate of an antibody to the macromolecular antigen and an enzyme for the substrate. PA1 (a) applying said sample on a substrate layer containing a non-diffusible substrate for forming a diffusible material in the presence of said enzyme, said non-diffusible substrate being a pulverized insoluble polysaccharide; PA1 (b) allowing to migrate said diffusible material formed in said substrate layer into a reagent layer for detecting said diffusible material; and PA1 (c) measuring the amount of said diffusible material migrating into said reagent layer. PA1 (a) applying said sample on a substrate layer containing a non-diffusible substrate for forming a diffusible material in the presence of said enzyme, said non-diffusible substrate being a pulverized insoluble polysaccharide; PA1 (b) allowing to migrate said diffusible material formed in said substrate layer into a reagent layer for detecting said diffusible material; and PA1 (c) measuring the amount of said diffusible material migrating into said reagent layer.
The antigen supplied by spotting onto the analysis element binds to the antibody-enzyme conjugate through a competitive reaction with the reaction of the polymerized antigen. The complex of antigen-antibody-enzyme reacts with the water-insoluble high polymer substrate to form a soluble lower molecular weight product. On the other hand, the complex of polymerized antigen and enzyme-labelled antibody formed by the binding with the polymerized antigen cannot exhibit the enzymatic activity to the high polymer substrate. Accordingly, as the quantity of the antigen in the sample is increased, the product produced by the enzymatic reaction increases. This product is allowed to diffuse into a detection layer where the quantity of the product is determined by measuring the optical density of an absorption resulted by the colored chemical group, to make it possible to analyze the antigen in the sample quantitatively.
The immunoassay element disclosed in Unexamined Japanese Patent Publication No. 295466/1991 (corresponding to U.S. Ser. No. 07/684,283 and EP 0451848A2) is an improvement of the aforementioned immunoassay element. This immunoassay element has a reagent layer containing a fragmenting enzyme for further fragmenting the decomposition product by the reaction of labelling enzyme, so that the fragmented lower molecular weight product is detected for further sensitization of the element.
When the analyte or ligand is a macromolecular antigen, the immunoassay element described in the specification of Japanese patent Appln. No. 248711/1990 (corresponding to U.S. patent application Ser. No. 07/763,198) may be used. This prior art element has the following two components either in a same layer or in different layers:
Likewise to the immunoassay element described in the specification of Unexamined Japanese Patent Publication No. 295466/1991 a reagent layer containing a fragmenting enzyme for further fragmenting the decomposition product by the action of labelling enzyme is provided so that the fragmented product having a lower molecular weight is detected to improve the sensitivity.
Anyway, it is desirous that the activity of the labelling enzyme to the high polymer substrate is sufficiently high. However, it is considered detrimental to use a water-insoluble high polymer substrate as the substrate for the enzyme, since such a sustrate is generally a macromolecular weight compound.